Chromatin modification of imprinted H19 gene in mammalian spermatozoa

The allele-specific epigenetic markings of endogenously imprinted genes in placental mammals occur during gametogenesis. The identification of the molecular nature of gametic imprints is the first step towards understanding the mechanistic basis of epigenesis in embryonic and adult somatic tissues. The specific question addressed in this work is whether the closely positioned but oppositely imprinted insulin-like growth factor 2 (IGF 2) and H19 genes, which have similar temporal regulation during development, differ in chromatin structure in mammalian spermatozoa. During terminal differentiation of mammalian spermatozoa, about 3–15% of the haploid genome retains a quasisomatic-type chromatin structure, whereas the remaining genomes interact with protamines that are further cross-linked by -S-S- bridges. Micrococcal nuclease (MNase) and DNase I digestions of human (HSN) and porcine sperm nuclei (PSN) showed that the IGF 2 gene in both types of nuclei retained somatic-type nucleosomes that were close-packed with a periodicity of 150 bp. However, the H19 gene in both species was predominantly organised by unique structural repeats, which were 650–674 bp in PSN and 438–522 bp in HSN, condensing at least 20 kb of chromatin. These results, together with previous studies, suggest that epigenetic chromatin modification leading to preferential condensation of the paternal H19 allele in embryonic tissues is already present in the germ cells. Mol. Reprod. Dev. 50:474–484, 1998. © 1998 Wiley-Liss, Inc.     

Galactose-1-phosphate uridylyltransferase. Purification of the enzyme and stereochemical course of each step of the double-displacement mechanism

A convenient new procedure for purifying galactose-1-phosphate uridylyltransferase from Escherichia coli is described. It departs from earlier methods by introducing the use of a Cibacron Blue-agarose (Bio-Rad Affi-Gel Blue) at an early stage. Purification is completed by ion-exchange chromatography using DEAE-Sephadex A-50. The procedure is substantially shorter than earlier methods and reproducibly yields enzyme of high specific activity suitable for use in structural work such as characterization of the intermediate uridylyl-enzyme. The first step of the galactose-1-P uridylyltransferase reaction is the transfer of the uridylyl group from UDP-glucose to N3 of a histidine residue in the enzyme to form the covalent uridylyl-enzyme and glucose-1-P. The uridylyl-enzyme intermediate then reacts in a second step with galactose-1-P to form UDP-galactose. The enzyme accepts (RP)-UDP alpha S-glucose as a good substrate, converting it to (RP)-UDP alpha S-galactose, i.e., with overall retention of configuration. In this paper we show that reaction of the enzyme with (RP)-[2-14C]UDP alpha S-glucose produces a [2-14C]uridylyl alpha S-enzyme that can be converted by base-catalyzed cyclization to (RP)-[2-14C]cUMPS. Inasmuch as cyclization must have proceeded with inversion of configuration at phosphorus, the corresponding configuration in the intermediate must have been the inverse of that in the substrate. Therefore, formation of uridylyl alpha S-enzyme from (RP)-UDP alpha S-glucose proceeds with inversion of configuration, and overall retention arises from inversion in each of the two steps. The results support the authenticity of the isolated uridylyl-enzyme as the true reaction intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)     

A juxtarenal myelolipoma in a cottontop marmoset (Saguinus oedipus): A case report

A myelolipoma was surgically removed from the abdomen of a 15-year-old female cottontop marmoset (Saguinus oedipus). Because of its close adherence to the right kidney, a unilateral nephrectomy was performed. The post-surgical recovery was uneventful. A myelolipoma is a circumscribed mass of bone marrow elements embedded in mature adipose tissue. These masses have been reported in the adrenal glands, paravertebral tissue, intrathoracic tissues, and mesentery of man. A similar condition has been observed in the liver and spleen of both captive and wild felidae. This report documents the first observation of this tumor-like condition in nonhuman primates with the possible exception of a subcutaneous myeloliposarcoma in a potto.     

Linking Habitat Restoration to Meaningful Units of Animal Demography

To restore habitat is to restore the functional aspects of a place where one or more species are intended to live. However, habitat restored with the right structural elements can still fail to support the species due to insufficient space and spatial contiguity (e.g., movement corridors) with nearby habitat. The spatial extent of habitat determines its capacity to support various demographic units, such as a breeding pair of individuals, a true population, or a metapopulation. In scatter‐plots of published density estimates and their corresponding study area sizes, I found consistent patterns that appeared to represent spatial scale domains, in which subpopulations, populations, and multiple populations exist for each species. These spatial scale domains can help guide habitat restoration by indicating the minimum area needed to support a population. The effectiveness of restored habitat, considered alone or in combination with existing habitat, should be judged by the habitat's capacity to sustain a functioning population, which includes the population's membership in a metapopulation. Additionally, the evidence increasingly demonstrates that habitat must include sufficient space for the population(s) to periodically relocate as resources are depleted locally. The spatial extent of proposed habitat restoration can now be linked to the likely demographic unit of the species that can be supported there. I provide two examples of proposed habitat conservation and their likely effectiveness based on their spatial areas.     

Mutational analysis of Norrin-Frizzled4 recognition

Norrin and Frizzled4 (Fz4) function as a ligand-receptor pair to control vascular development in the retina and inner ear. In mice and humans, mutations in either of the corresponding genes lead to defects in vascular development. The present work is aimed at defining the sequence determinants of binding specificity between Norrin and the Fz4 amino-terminal ligand-binding domain (the "cysteine-rich domain" (CRD)). The principal conclusions are as follows: 1) Norrin binds to the Fz4 CRD and does not detectably bind to the 14 other mammalian Frizzled and secreted Frizzled-related protein CRDs; 2) Norrin and Xenopus Wnt8 recognize largely overlapping regions of the Fz4 CRD; 3) surface determinants on the Fz4 and Fz8 CRDs that allow Norrin to distinguish between these two CRDs reside within several small regions on one face of the CRD; 4) Norrin function depends critically on three pairs of cysteines that form the highly conserved trio of disulfide bonds shared among all cystine knot proteins, but the remaining two putative disulfide bonds are less important; 5) Norrin-CRD binding depends on a largely contiguous group of amino acids in the extended beta-sheet domain of Norrin that are predicted to face away from the interface between the two monomers in the Norrin homodimer; 6) Norrin-CRD binding is strongly modulated by interactions involving charged amino acid side chains; and 7) Norrin-CRD binding is enhanced approximately 10-fold by the addition of heparin. These observations are discussed in the context of Frizzled signaling and the structure and function of other cystine knot proteins.     

Synthesis and SAR of potent LXR agonists containing an indole pharmacophore

A novel series of 1H-indol-1-yl tertiary amine LXR agonists has been designed. Compounds from this series were potent agonists with good rat pharmacokinetic parameters. In addition, the crystal structure of an LXR agonist bound to LXRα will be disclosed.     

Co-Regulation of NF-κB and Inflammasome-Mediated Inflammatory Responses by Myxoma Virus Pyrin Domain-Containing Protein M013

NF-κB and inflammasomes both play central roles in orchestrating anti-pathogen responses by rapidly inducing a variety of early-response cytokines and chemokines following infection. Myxoma virus (MYXV), a pathogenic poxvirus of rabbits, encodes a member of the cellular pyrin domain (PYD) superfamily, called M013. The viral M013 protein was previously shown to bind host ASC-1 protein and inhibit the cellular inflammasome complex that regulates the activation and secretion of caspase 1-regulated cytokines such as IL-1β and IL-18. Here, we report that human THP-1 monocytic cells infected with a MYXV construct deleted for the M013L gene (vMyxM013-KO), in stark contrast to the parental MYXV, rapidly induce high levels of secreted pro-inflammatory cytokines like TNF, IL-6, and MCP-1, all of which are regulated by NF-κB. The induction of these NF-κB regulated cytokines following infection with vMyxM013-KO was also confirmed in vivo using THP-1 derived xenografts in NOD-SCID mice. vMyxM013-KO virus infection specifically induced the rapid phosphorylation of IKK and degradation of IκBα, which was followed by nuclear translocation of NF-κB/p65. Even in the absence of virus infection, transiently expressed M013 protein alone inhibited cellular NF-κB-mediated reporter gene expression and nuclear translocation of NF-κB/p65. Using protein/protein interaction analysis, we show that M013 protein also binds directly with cellular NF-κB1, suggesting a direct physical and functional linkage between NF-κB1 and ASC-1. We further demonstrate that inhibition of the inflammasome with a caspase-1 inhibitor did not prevent the induction of NF-κB regulated cytokines following infection with vMyxM013-KO virus, but did block the activation of IL-1β. Thus, the poxviral M013 inhibitor exerts a dual immuno-subversive role in the simultaneous co-regulation of both the cellular inflammasome complex and NF-κB-mediated pro-inflammatory responses.